Our goal is to understand the macromolecular interactions maintaining long range DNA folding in chromosomes, and we propose to focus on two areas of bacterial chromosome structure. The first involves DNA supercoiling and DNA gyrase. Oxolinic acid specifically inhibits gyrase, introducing DNA breaks at the sites of gyrase action. We will determine if during the cell cycle gyrase changes location relative to markers like integrated bacteriophage lambda. If it does, then the drug-induced fragments containing phage DNA would change size. We will also determine if gyrase is clustered around the replication fork by seeing if pulse-labeled DNA is concentrated in small fragments. The second area utilizes the nonintegrative attachment of plasmid DNA to the folded chromosome to prepare DNA-protein-RNA complexes which may be involved in DNA folding. Folded chromosome-plasmid DNA complexes will be isolated, and the types of interactions responsible for maintaining them will be characterized. Subcomplexes of plasmid DNA attached to chromosomal fragments will be generated by shearing to break chromosomal DNA, but not plasmid DNA. The components of these subcomplexes will be analyzed to determine if specific proteins are present.